Af 267 na

Animals were deeply anesthetized with ketamine/xylazine and perfused with 4% PFA/PBS. Brains were removed and postfixed in 4% PFA/PBS overnight at 4°C, then cryopreserved with 30% sucrose. Sections (20 µm) were cut using a Leica cryostat, and mounted onto charged slides. Sections were permeabilized with 0.5% triton in PBS for 10 min and blocked with 1% BSA and 5% donkey serum in PBS for 1 hr at room temperature. Primary and secondary antibodies were prepared in 1% BSA. Sections were incubated with primary antibodies overnight at 4°C in a humidified chamber. Antibodies used were: Ki67 (Abcam 15580, RRID:AB_443209, 1/500), anti-p75 (R&D AF367, RRID:AB_2152638, 1/500), anti-p75 (Millipore MAB365, RRID:AB_2152788, 1/1000), anti-proNT-3 (R&D AF3056, RRID:AB_2154250, 1/200), anti-NT-3 (R&D AF-267-NA, RRID:AB_354434, 1/200), anti-BrdU (Millipore 05–633, RRID:AB_309861, 1/50). All secondary antibodies were diluted 1:1000, and incubated for 1 hr at RT. Nuclei were labeled with 1 µg/ml Dapi/PBS for 1 min at RT or 1 mM Draq5 (BioStatus DR-50200) for 30 min and mounted using Prolong Gold (Life Technologies P36931). Controls for immunostaining included incubation with secondary antibodies in the absence of primary antibodies. The proNT3 antibody was tested for specificity on sections from Ntf3-/- mice compared to WT from P0 pups, since the Ntf3 -/- mice die after birth.

Protein expression in adipose tissue was measured by immunoblotting as we described^{69 (link),76 (link),77 (link)}. Fat tissues were homogenized in a modified radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1% protease inhibitor mixture and 1% phosphatase inhibitor mixture (Sigma-Aldrich, St. Louis, MO). Tissue lysates were resolved by SDS-PAGE. Proteins on the gels were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA), which were then blocked, washed, and incubated with various primary antibodies, followed by Alexa Fluor 680-conjugated secondary antibodies (Life Science Technologies). The blots were developed with a Li-COR Imager System and analyzed with Li-COR Image Studio Software (version 2.1, Li-COR Biosciences, Lincoln, NE). The following primary antibodies were used: UCP1 (1:1000, Abcam, ab23841), TH (1:1000; AB152, EMD Millipore, Temecula, CA), NT-3 (1:1000, AF-267-NA, R&D Systems), pHSL (1:1000, 4126S, Cell Signaling Technology), HSL (1:1000, 4107S, Cell Signaling Technology), and α-tubulin (1:1000, Advanced BioChemicals, ABCENT4777). The antibody information is listed in Supplementary Table 1.
Tissue and serum NT-3 content was measured by ELISA (ABCE-EL-M2438, Advanced BioChemicals, Lawrenceville, GA).